1. Oubain-sensitive HCO 3 - secretion & acid absorption by the marine teleost fish play a role in osmoregulation M. Grosell & J. Genz

3. Study Goals Determine the contribution of endogenous metabolic CO 2 vs extracellular HCO 3 - as sources of secreted HCO 3 - Determine role of carbonic anhydrase (CA) in hydrating endogenous CO 2 Determine fate of liberated H + from CA-mediated hydration reaction

4. Endogenous CO 2 is hydrated in a reaction regulated by carbonic anhydrase (CA). The resulting HCO 3 - is actively transported across the apical membrane via Cl - /HCO 3 - exchange (AE). This is driven by the + potential inside the cell set up by NKA. Fig. 11 Cellular mechanisms in intestinal epithelial transport of HCO 3 -

6. Results Resting tissue –Viable and stable for >5 h –secretion rates are between 0.3-0.5 umol/cm 2 /h –TEP = -20 mV –Conductance = 10 mS/cm 2 HCO 3 - secretion decreases when deprived of O 2 Secretion is strongly temperature dependant

7. Extracellular HCO 3 - vs. Endogenous CO 2 HEPES vs. 5 mM HCO 3 - saline When exposed to HCO 3 - /CO 2, base secretion increased twofold Figure 4

8. Hydration of endogenous CO 2 accounts for ~50% of total HCO 3 - secretion Percentage contributed by hydrated endogenous CO 2 is species-dependant –30-60% CO 2 in European flounder (Grosell et al 2001) and goby (Dixon & Loretz 1986) –100% extracellular HCO 3 - in Japanese eel (Ando & Subramanyam 1990) % of endogenous CO 2

9. Role of CA Pharmacological test - Etoxzolamide Figure 5

10. What happens to H + ? Must be removed to –Maintain constant intracellular pH –Prevent reversal of CA-catalyzed hydration

11. Possible pathways for removal of H + across the basolateral membrane is either Na + /H + exchange (NHE) or an H + pump. Na + dependence (Fig. 8) suggests NHE.

12. Altering serosal pH pH% Reduction 7.433 7.044 6.647

13. Directly measure H + transport

14. Pharmacology Not inhibited by EIPA or Amiloride –Does not discount NHE Significant reduction when exposed to oubain (NKA inhibitor)

15. Net acid/base flux

16. Ca:Mg ratio in precipitate Salinity (ppt)93350 Ca 2+ (mmol/g)-0.20 ± 0.140.44 ± 0.140.65 ± 0.04 Mg 2+ (mmol/g)-0.02 ± 0.024.13 ± 1.960.47 ± 0.02 Ca:Mg ratio6.54 ± 1.950.16 ± 0.031.36 ± 0.08 Intestinal Rectal Salinity (ppt)93350 Ca 2+ (mmol/g)0.15 ± 0.050.08 ± 0.030.14 ± 0.07 Mg 2+ (mmol/g)0.03 ± 0.010.13 ± 0.030.19 ± 0.03 Ca:Mg ratio4.92 ± 1.23-2.36 ± 3.150.45 ± 0.29

17. Further Discussion Possibility of global impact of precipitates Whole-animal acid/base balance This process is similar to mammalian pancreatic secretion –Could intestinal HCO 3 - secretion be common to all vertebrates?